189management of patients with esophageal adenocarcinoma. The
ability for cfDNA and mutations within to be detected consistently,
early, and unambiguously makes it an excellent tool for surveillance
and management of ongoing treatment.
There are additional challenges in regard to what method of
liquid biopsy is the best to use. To some degree, this will be dic-
tated by patient-specific conditions, but ongoing research will be
needed to determine precisely what methods provide the most use-
ful information to clinicians for patient management. In general,
however, use of next generation sequencing on obtained nucleic
acids is recommended for sensitivity to tumor heterogeneity, but
also to detect low level cancer signals where benign cells may be
the majority signal identifiable, as in crude cell isolates from blood
or exosome-derived RNA. Similarly, sensitive methods should be
used for the detection of proteins or other substances for the same
reasons.
The following protocol will cover methods for liquid biopsy
concerned with isolation of nucleic acids, but can serve as a basis
for the extraction of other biomolecules for tumor assessment.
Likewise, this protocol is concerned with deriving cancer cell DNA
from blood. In addition, other appropriate body fluids (such as
saliva, urine, stool, or cerebrospinal fluid) could be used depending
on the cancer of origin, or location of metastases [ 3 – 5 , 8 – 11 ].
Collection of circulating tumor DNA (ctDNA) can be achieved
from numerous sources. Obtaining cell-free DNA from serum or
plasma has the advantage of removing excessive signal from lym-
phocytes and other wild-type DNA from benign cells from the
sample, without needing to undertake cell sorting or cell selection,
which are themselves a highly specialized set of methods undergo-
ing continuous refinement (beyond the scope of this protocol).
With that said, the DNA derived from serum and plasma is typi-
cally highly fragmented, so care should be taken when using meth-
ods of purification that small size DNA fragments are not lost.
Both serum and plasma have been able to be used for detection of
ctDNA, but the potential for leakage of benign DNA from cells
during the extended handling and coagulation of serum make it a
potentially less attractive option [ 5 , 8 , 12 ]. As such, this method
will concern itself with the collection of plasma rather than serum,
but serum can be used interchangeably with the method.2 Materials
Prior to commencing the method, the following items should be
available:- Pipettes.
- Centrifuge tubes, ranging from 10 to 1 mL, depending on
volumes needed.
Cell-Free Plasma and Exosome-Associated DNA